Screening, Cloning And Expression Of Bacterial Thermophilic β-glucosidase

β-glucosidase (EC.3.2.1.21) is an important hydrolytic enzyme in cellulase system, which was widely used in industry, agriculture, medicine and other areas. Thermophilicβ-glucosidase has broad application prospects in food fermentation, ethanol fuel and other areas, because of its advantage in accelerating reaction and reducing energy consumption, etc. This study aims at exploring a thermophilicβ-glucosidase which could cooperate with diastatic enzyme to enhance the utilization ratio of raw materials and productivity of ethanol during the process of ethanol fuel production.A thermophilic bacteria strain (F157043A) with relatively high capability of producingβ-glucosidase was obtained and was identified as Bacillus licheniformis by method of molecular biological identification, physiological and biochemical property analysis. Theβ-glucosidase in crude extract was characterized and bglH was cloned into E. coli and successfully expressed.1. Based on Culture Collection Center of Industrial Microorganisms in Jiangnan Uninversity (CICIM-CU), 20 out of 1323 bacteria strains were isolated with relatively high capability of producing thermophilicβ-glucosidase. 5 strains were identified as Geobacillus sp. and others were identified as Bacillus licheniformis by 16S rDNA sequencing analysis. Two bacteria strains (170-28-2 Geobacillus sp. and F157043A Bacillus licheniformis) with higher capablility of producingβ-glucosidase were obtained during secondary screening. After preliminarily characterizing theβ-glucosidase in crude extract with two strains, the bacteria F157043A, theβ-glucosidase of which had better thermophily and wider range of pH stability, was designated as the object for further study.2. After study on morphological, physiological and biochemical properties, the strain of F157043A was further identified as Bacillus licheniformis.3. The basic properties of the enzyme in crude extract for Bacillus licheniformis (F157043A) were studied. It was optimally active at 55℃and pH 9.0; it retained 80% of its optimal activity at 60℃and was stable in the pH range 6.0-9.0; Xylose, glucose and fructose evidently inhibited its activity; the presence of metal ions such as Mg²⁺, Co²⁺, Mn²⁺ positively influenced its activity, but the activity was greatly inhibited in the presence of Cu²⁺, Zn²⁺, Ca²⁺ and SDS;β-glucosidase activity was inhibited in various degrees by different concentrations of alcohol.4. The 1410 bp gene ofβ-glucosidase(bglH) was cloned from Bacillus licheniformis (F157043A) and was linked into the expression vector pET-20b(+). The recombinant plasmid was transformed into E. coli BL21(DE3). After induced by IPTG, recombinant enzyme with an estimated molecular weight of 53 kDa on SDS-page was found to be expressed. The specificβ-glucosidase activity in crude extract for the recombinant strain and the control were 0.14 U/mg and 0.04 U/mg at 60℃respectively. After optimization for cultivation conditions, the specific enzyme activity of recombinant strain was enhanc to 0.54 U/mg.5. Aiming at the phenomenon of E.coli BL21(DE3)'s backgroundβ-glucosidase activity during the experiment, which was not mentioned in the published references, several experiments were designed and prelimilarily clarified the positive affect of IPTG on the formation of P-β-glucosidaseA in E. coli BL21(DE3).